Pigeon is a beautiful domestic bird. The relationship between pigeons and man is very ancient. In the ancient period, some pigeons were trained up for carrying mail to inaccessible places, some pigeons were used for the satisfaction of God and Goddesses, and the rest of the pigeons were used as an excellent source of palatable meat. Now pigeon is worldwide famous for their different attractive feather colors and melodious voices, and are considered an ambassador of peace (Shantir Dut). As a tonic diet, pigeon meat is widely used by convalescent patients in Bangladesh and China (Saikat et al., 2020; Kabir et al., 2015). Many years ago, the maximum housewife in maximum villages and urban areas rearing at least 10-20 pigeons in a house made of wood or bamboo strips, placed in a shaded place in a house compound or building ledges because of getting 13 pairs squabs from one pair pigeon in 12 months, less feeding cost, less number of infectious diseases, less treatment cost, easy domesticating, and rearing processes, etc (Prabakaran et al., 2020). 

Now high-breed pigeons are rearing commercially in many countries of the world including Bangladesh (Black section, Yellow section, Yellow karnew, White short phase, Yellow dyne phase, White bokhra, Black bokhra, Brown bokhra, Blue bokhra, Danish can, Stick viena, White lakkha etc) because of its excellent bred characteristics (especially beautiness), high profit and increased demand (Kabir et al., 2021). In Bangladesh, besides indigenous breeds, a significant number of commercial pigeon farms are developing day by day in some districts named Pabna, Nator, Rongpur, Mymensingh, Dhaka, Norsingdhi, Chattogram, Coxs Bazaar, Jessor, and Potuakhali and in most of the cases, these are being reared by unemployed peoples (Asaduzzaman et al., 2009).  Mentionable that, when in an area, most of the farmers are being financially benefitted through the year, someone causing huge economic loss when a huge number of pigeons either high-breed or indigenous breeds die on own farm being infected with a highly fatal viral disease named Newcastle disease. 

Newcastle disease virus is a single-stranded RNA virus that is a virulent strain of avian paramyxovirus type 1 (APMV-1). Newcastle disease (ND) affects numerous domestic and wild bird species (Cardenas et al., 2013). Newcastle disease is the most important viral poultry illness worldwide, including in emerging areas (Sharif et al., 2014).  In Africa and Asia, ND is a big reason why industrial and village poultry production cant grow as much as it could. Depending on the pathotype of the virus, NDV infections in chickens can be harmless or kill them quickly (Alexander et al., 2003). There is no good way to treat NDV, but using preventative vaccines and keeping things clean can help stop outbreaks. It has been said that the only way to protect against endemic ND is to get vaccinated (Nwanta et al., 2006). An epidemic spreading of Newcastle disease usually occurs in the susceptible non-vaccinated birds in a local farming area when these remain non-vaccinated, a huge number of commercial hen farms and pigeon farms are developed closely or haphazardly and maintained unhygienically. 

The infected pigeon dies usually due to hypovolemic shock and respiratory failure, and disease spreading to the surrounding pigeon farms usually occurs through contaminated (by nasal droplets) air. Of this aerosol-spreading characteristic of the disease, Newcastle disease is a great threat to the development of sustainable and profitable high-bred pigeon farms, also indigenous-breed pigeons in developing Bangladesh. Mentionable rehearsed works on pigeon Newcastle disease have not been done in Bangladesh. A study was carried out to determine the antibody against Newcastle disease and also determine serum protein levels in pigeons in Chattogram metropolitan area. The antibody titer in NDV pigeons was done by HI test. It was found that considerable amounts of pigeons carried less than protective HI titer and were susceptible to NDV infection. The pigeons were not regularly vaccinated against NDV. The antibody titer which is found may be due to some other paramyxovirus that has antigenic resemblance with NDV. Total protein in pigeons carried less than normal range suggesting carries worm infection or proteinemia. 

For satisfactory prevention and control of pigeon Newcastle disease incidence in Bangladesh, this study should be done by increasing sample size and attempt should be taken for virus isolation. Virus can be characterized by molecular techniques and compared with virus isolating from chicken to see a diversity of antigenicity of NDV. Therefore, the main objectives of this research were to the determination of antibody against Newcastle disease in pigeons; determine various risk factors for contributing to NDV infection, and determination of serum protein levels in pigeons in the Chattogram area in Bangladesh. 

Ethical consideration

The ethical approval of the current research was taken from the Departmrnt of Microbiology Gono Bishwabidyalay, Dhaka-1344, Bangladesh. 

Study area selection

This research was conducted by the Department of Microbiology, Gono Bishwabidyalay, Savar Dhaka, and Department of Microbiology and Veterinary Public Health, Faculty of Veterinary Medicine, Chattogram Veterinary and Animal Sciences University (CVASU), Khulshi, Chattogram period of 2019. A total of 90 different pigeons (local, giribaj, siraji) were selected for this research, and samples were collected from different areas of the Chattagram area, Bangladesh.  

Blood collection

Blood was collected from different pigeon species by using an antiseptic swab, a sterile syringe, and a vial containing EDTA as an anticoagulant. Blood was collected from the wing vein in the case of avian species, from the jugular vein in the case of mammalian species, and the brachial vein in the case of humans. After collection blood was transported to the laboratory for the preparation of 1% RBC (Manual 2004).

Preparation of 1% RBC

Blood from each species was taken and put into a falcon tube that could be spun in a centrifuge. The tube was then filled with PBS, and it was spun for 10 minutes at 1500 RPM. The supernatant was thrown away by washing the Pasteur pipette while being careful not to move the RBC pellet. The step of spinning three times was done three times. After being washed three times and put in a centrifuge, the cells now look like a ball. Next, the packed cell volume (PCV) was measured, and a 1% solution of red blood cells was made for storage by mixing 1 ml of packed cells with 99 ml of PBS (Manual 2004).

Preparation of 4 HA unit virus

In the haemagglutinin titration, one HA unit is the smallest amount of virus that will cause the RBC to stick together. To find out how much virus was present, a HA test was done with 1% chicken RBC and a two-fold dilution of the virus. One haemagglutinating unit is in the last well of the 96-well plate that shows the full haemagglutination with 1% chicken RBC. Then, the well with 4 HA units was found, and the dilution factor was worked out. To make a suspension of 4 HA units of the virus, 1 ml of the virus was mixed with the right amount of diluent.

Hemagglutination (HA) test

The procedure of HI test was followed as per method described by Anon. (1971) and divided into two parts:

microplate HA test to determine HA units (4 Units/25μl) and microplate HI test. An amount of 50 µl of PBS was dispensed into each well of the microwell plate up to column 7. Then 50 ml of test samples (virus) were placed in the first well of each row of column 1. By using a multichannel pipette a two-fold serial dilution was carried out across the plate until column 6. Then 50 ml of 1% red blood cells of tested species were added in each well including column 7. The wells in this column 7 are control wells that contain only PBS and RBC. The sides of the plate were gently tapped to mix and a cover on the plate was placed and allowed the plate to stand at three different temperatures (e.g Chilling, Room temperature, and incubation temperature). Results were taken every 5 minutes after the settlement of RBC in the control row and recorded in a data sheet.

Hemagglutiantion Inhibition test (HI)

An amount of 50 µl of PBS was dispensed into each well of the microwell plate. Then 50 µl of test sera samples were placed in the first well of each row of column 1. By using a multichannel pipette a two-fold serial dilution was carried out across the plate until column 10. Then 50 µl of 4 HAU was added in each well up to column 11 and left for 30 minutes at room temperature. 50 µl of 1% red blood cells was added to each well of the microtitre plate. The wells in this column 12 are control wells that contain only PBS and RBC. The sides of the plate were gently tapped tp mix and a cover on the plate was placed and allowed the plate to stand at three different temperatures (e.g Chilling, Room temperature, and incubation temperature). Results were taken every 5 minutes after the settlement of RBC in the control row and recorded in a data sheet.

Total protein (TP) determination

In the present work, the reagent kit was intended to quantitatively determine Total Protein in serum/ plasma. Briley, 10 µl serum samples were mixed with 1 ml biuret reagent, then 10 µl standard was mixed with 1 ml biuret reagent, while blank was mixed with 1 ml biuret reagent.  After mixing well, all test tubes were incubated for 5 minutes at 25°C. Next, a measure of absorbance of the serum sample (T) and standard (S) against the reagent blank was conducted after 60 minutes. The total protein determination was done by use of a Biochemistry Analyzer. After completing the absorbance measure, the final total protein concentration (gm/dl) was calculated by use of the formula:

Abs. T/Abs. S* 6. 

Where Abs. T is the absorbance test of the sample, Abs.S is the absorbance standard 

Statistical Analysis

Data from different sampling sites were entered in Excel sheets (Microsoft Excel) and analyzed by using SPSS version 21. The results of all data are summarized by descriptive analysis. The chi-square test was also applied to measure the significant difference in several sampling areas. Confidence intervals were determined using the Binomial exact test, with a significance level set at less than 0.05 to establish statistical significance.

HI titer against Newcastle Disease Virus in the pigeon (according to different age groups for HI titer)

Antibody titer against Newcastle Disease virus was analyzed according to the different age groups of pigeons by X2 test. There was significant variation in different aged pigeons. The total number of pigeons was ninety (90). The groups were classified according to below 6 months, more than 6 months but less than 1 year, and above 1 year. In pigeons less than 6 months of age it was shown about 73% (n=22) were HI titer positive whereas in birds in a group where age was more than 6 months but less than 1 year  56% (n=32) were HI negative. Few birds (n=3) that belonged to the class for more than 1 year were 100% HI titer positive (Table 1).

Table 1: HI titer against Newcastle Disease Virus in the pigeon (according to different age groups).

Antibody titer against Newcastle Disease virus was analyzed according to the sex of the pigeon by X2 test. There was no significant variation among different sexes. In male and female pigeons both groups had almost equal numbers of pigeons that were HI titer positive. In both male and female groups, HI-positive pigeons were slightly higher than HI-titer-negative birds. The percentage of HI titer-positive pigeons in females and males was 51.7% and 57.4% respectively (Table 2).

Table 2: HI titer against Newcastle Disease Virus in the pigeon (according to Sex).

HI titre against Newcastle Disease Virus in the pigeon (according to different breeds of pigeon)

Antibody titer against Newcastle Disease virus was analyzed according to different breeds of pigeons by X2 test. The breeds were Giribaj, King, Local, Owl, and Siraji. There was significant variation among the different breeds. Most of the pigeons (n=68) belonged to the local breed and the antibody titer against Newcastle Disease Virus in the local breed was almost equal (Table 3 and Fig. 1).

Table 3: HI titer against Newcastle Disease Virus in the pigeon (according to different breeds of pigeon).

Breed	HI titer negative in percentage	HI titer positive in percentage	X2 value
Giribaj	50.0 (n=4)	50.0 (n=4)	0.043
King	0 (n=0)	100 (n=6)
Local	51.5 (n=35)	48.53 (n=33)
Owl	0 (n=0)	100 (n=3)
Siraji	20 (n=1)	80 (n=4)

Fig. 1: HI titer against Newcastle Disease Virus in the pigeon (based on clinical signs of ND Infection).

Antibody titer against Newcastle Disease virus was analyzed based on clinical signs of ND infection in pigeons by X2 test. There was significant variation among NDV-infected and non-infected pigeons.  In the case of ND noninfected pigeons, there are almost equal percentages of HI titer positive and negative birds whereas in the case of ND infected most of the pigeons (80.8%) were HI titer positive (Table 4).

Table 4: HI titer against Newcastle Disease Virus in pigeons (based on clinical signs of ND infection).

Presence/absence  of clinical signs of ND Infection	HI titer negative in percentage	HI titre positive in percentage	X2 value
ND-non infected Pigeon	54.69 (n=35)	45.31(n=29)	0.002
ND-infected pigeon	19.23 (n=5)	80.8 (n=21)

Comparison of HI titer between total serum proteins

A paired t-test was done between HI titer and total serum protein. There was a significant (t<0.0) difference among the groups. In the case of HI titer mean was about 23 titer and the standard error was 0.28. In the case of total serum protein mean was 4.45 and the standard error was 0.19 (Table 5).

The Haemagglutination Inhibition (HI) test was used to find out how many people had antibodies against the Newcastle Disease (ND) virus and how strong those antibodies were. The amount of HI in each bird was measured and written as a log2 number, and the mean for each species was found. This study was carried out to evaluate the seroconversion of Newcastle Disease Virus in pigeons in the surroundings of Chattogram Area Bangladesh and pigeons that were brought from Teaching Veterinary Hospital (TVH) for treatment purposes in Chattogram Veterinary and Animal Sciences University (CVASU). The mean HI titer (n=90) was log based 22.95 with a standard error of 0.28 which means lower than ≤24 indicating negative results according to the OIE standard (Manual, 2004).  In our study we evaluated the titer of NDV according to different age groups, it was revealed 52% of pigeons possess NDV antibody titer which has a resemblance with 51% (Elina et al., 2017). NDV can cause infection in the pigeon (Columba livia) as well as other members of the Columbia family have been documented by several authors (Biancifiori et al., 1983 & Kapczynski et al., 2006) from various sources like diseased chickens, and domesticated or feral pigeons (Toro et al., 2005). NDV can survive in the feces for long period, therefore in absence of any signs outbreaks can occur as a result of direct contamination of untreated food the pigeon (Toro et al., 2005 & Alexander et al., 1984). 

The risk of the transmission of NDV from poultry to pigeons (n=100). Antibodies to NDV were detected in 17%, while 83% were non-detectable (NDV) antibodies. Studies have shown that pigeons are susceptible to NDV. This might be due to occasional visits of these birds to the poultry premises where they could have contracted the virus from materials from poultry houses where ND infection is endemic. Wild pigeons can tolerate certain disease agents as well as a reservoir for spreading many parasitic and microbial species to other types of birds especially (Poultry), without displaying gross pathological changes and clinical signs of sickness (Barwary et al., 2015). In the case of NDV young birds are more susceptible than adults (Barwary et al., 2015). No natural infection in pigeons was observed before 1971 however, an outbreak of epidemic killed many pigeons in Europe between 1971-73 (Kommers et al., 2002). In our study, we evaluated antibody titer in apparently ND-infected pigeons and not ND-infected pigeons by X2 test. There was a significant difference (P<0.005) between the titer of the two groups. In ND infected (apparently) seropositive was about 81% and about 19% were seronegative birds.  However, in ND noninfected groups there was an almost equal percentage of seropositive and seronegative birds. In seropositive, the percentage was 45.31, and in seronegative was 54.69. It might be due to the shedding of the NDV virus from an infected pigeon and getting into a noninfected pigeon and giving rise to seroconversion. In one study in Tanzania, it was found that 10% of sera samples were positive for NDV in domesticated pigeons and it might be due to mixing with (Wambura et al., 2010). HI, titer was evaluated among different breeds of pigeon. There was a significant difference among different breeds (p<0.005) by the X2 test. In local and breed Giribaj almost equal percentage of pigeon HI positive and HI negative antibody titers but in the case of Siraji (80%) and King (100%) was HI titer positive against NDV. This might be due to the small size of the population. Measuring serum protein is an important diagnostic tool for the uncovering, diagnosis, and monitoring of various diseases and pathological conditions. Immunoglobulin is a major component of serum protein which can be reflected in the immune response. Most of the serum proteins are produced and secreted from the hepatocyte. Notably, the exception is immunoglobulin which is produced by plasma cells (Eckersall et al., 2008). Moreover, some studies show that nonhepatic tissue like the intestine, lung, adipose tissue and can be capable to synthesize some serum proteins for specific functions (Iliev et al., 2018). In vivo anabolism and catabolism of protein and status of immunity are usually reflected upon serum protein level. Increased level of albumin and globulin is an indication of an enhanced immune system (Zhang et al., 2013).

Plasma protein is made up of a heterogeneous mixture of proteins having different structural and functional attributes. The principal protein comprises albumin, globulin, fibrinogen, nucleoprotein, and conjugated proteins such as lipoprotein and zero-mucoid protein (Taylor et al., 2005).  In our study, the mean serum protein is 4.46 gm/dl with a standard error of 0.19. In one study evaluating serum total protein in young racing pigeons was revealed that adult pigeons contain 3.81 ± 0.03 gm/dl in 30-week-old pigeons (Gamal et al., 2014). Most of the proteins in birds plasma are albumin, which makes up about 40–60% of all proteins (Oladele et al., 2008). If a disease changes the number of proteins in plasma, it can affect its nutritive, physicochemical, or transportive functions (Taylor et al., 2005). Furthermore, According to our findings, Authors realized that regular vaccination of pigeons in NDV is really important because non vaccinated pigeon can easily transmitted NDV from pigeons to another animals which finally effect on human beings. Consequently, due to short research period and less sample size we could not pointout exact seroprevalence of NDV in pigeon farms. Finally, authors think that another research is needed in future where more sample will be considered for NDV detection from Chattagram region, Bangladesh. 

M.A.T.; and M.R.A. designed, conceptualization, writing the manuscript. M.A.H.; and Z.A.S. checked plagiarism and writing review and correction. M.A.T.; A.R.; and M.A.H. contributed in data analysis. M.S.M.; A.S.M.M.; and M.A.H. finally checked the manuscript and editing. All authors who are involved in this research read and approved the manuscript for publication. 

All raw data are available, if required data can be provided to the editors when we are asked for it.

The antibody titer in NDV pigeons was done by HI test. It was found that considerable amounts of pigeons carried less than protective HI titer and were susceptible to NDV infection. The pigeons were not regularly vaccinated against NDV. The antibody titer which is found may be due to some other paramyxovirus that has antigenic resemblance with NDV. Total protein in pigeons carried less than the normal range suggesting carries worm infection or proteinemia.  This study should be done by increasing the sample size and an attempt should be taken for virus isolation. Viruses can be characterized by molecular techniques and compared with virus isolating from chicken to see the diversity of antigenicity of NDV.

The authors would like to thank the market people for giving their support to collect samples. The authors also thank the Department of Microbiology, Gono Bishwabidyalay for their laboratory facilities. 

The authors declare that there is no conflict of interest.